Research is focused on studying the events involved in HIV-1 fusion, the development of fusion inhibitors directed against gp41 and the development of monoclonal antibodies with broadly neutralizing activity that are directed against gp41. The HIV-1 Envelope (Env) proteins that mediate membrane fusion represent a major target for the development of new AIDS therapies. Three classes of Env-mediated membrane fusion inhibitors have been described that specifically target the pre-hairpin intermediate conformation of gp41. Class 2 inhibitors bind to the C-terminal heptad repeat (C-HR) of gp41. The single example of a class 3 inhibitor targets the trimeric N-terminal heptad repeat (N-HR) of gp41 and has been postulated to sequestrate the N-HR of the pre-hairpin intermediate through the formation of fusion incompetent heterotrimers. We have now shown that NCCG-gp41, a class 2 inhibitor, and N36Mut(e,g), a class 3 inhibitor, synergistically inhibit Env-mediated membrane fusion for several representative HIV-1 strains (X4 and R5) in both a cell fusion assay (with membrane-bound CD4) and an Env-pseudotyped virus neutralization assay. We have also succeeded in obtaining a monoclonal Fab (Fab 3674) selected from a human non-immune phage library by panning against the chimeric construct NCCG-gp41 (that comprises an exposed coiled-coil trimer of gp41 N-helices fused in helical phase onto the minimal thermostable ectodomain of gp41) that effectively neutralizes diverse laboratory-adapted B-strains of HIV-1 and primary isolates of subtypes A, B and C in an Env-pseudotyped virus neutralization assay, albeit with reduced potency (25x) compared to 2F5 and 4E10. Alanine scanning mutagenesis maps a novel epitope to a shallow groove on the N-helices of gp41 that is exposed between two C-helices in the fusogenic six-helix bundle conformation of gp41. We have shown that bivalent Fab 3674 and the C34 peptide (a potent fusion inhibitor derived from the C-helix of gp41) act at similar stages of the fusion reaction and to neutralize HIV-1 synergistically, providing additional evidence that the epitope of Fab 3674 is new and distinct from the binding site of C34.More recently we have shown that the class 3 inhibitor N36Mut(e,g) prolongs the temporal window during which the virus is susceptible to neutralization by the bivalent Fab-3674, and that bivalent Fab-3674 and N36Mut(e,g) neutralize HXB2 and SF-162 strains of HIV-1, as well as diverse primary B and C clade HIV-1 strains, synergistically in a Env-pseudotyped virus neutralization assay. N36Mut(e,g) also rescues neutralizing activity of the monovalent Fab-3674 against resistant HIV-1 strains and renders a series of related non-neutralizing Fabs neutralizing. Moreover, N36Mut(e,g) exhibits the same effects on the broadly neutralizing 2F5 and 4E10 monoclonal antibodies directed against the membrane proximal extended region of gp41. Very recently, we have subjected Fab 3674 to affinity maturation against the NCCG-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtypes B and C HIV-1 reference panels. The parental Fab 3674 is 10-20 fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format (50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format (120 kDa) across the subtypes B and C reference panels. We have now solved crystal structures of various neutralizing and non-neutralizing Fabs to two gp41 pre-hairpin intermediate mimetics. These studies provide novel structural insights into immunogen design.